Hyperthermophiles are microorganisms which grow optimally at temperatures of 80° C. and above (Huber et al., J. Biotechnol. 64, 39-52 (1998)). A majority of these organisms fall under the newly identified domain, archaea (Woese et al., Proc. Nat. Acad. Sci USA 87, 4576-4579 (1990)). One of the most extensively studied hyperthermophilic archaea is Pyrococcus furiosus (Pfu). This microorganism has a growth optimum of 100° C. (Fiala et al., Arch. Microbiol. 145, 56-61 (1986)). It is a heterotrophic, anaerobic archaeon, and utilizes complex carbohydrates and peptides/proteins as carbon and energy sources (Adams et al., Adv. Protein Chem. 48, 101-180 (1996)). The metabolic end products are organic acids, alanine, CO2 and H2 (Kengen et al., Arch. Microbiol. 161, 168-175 (1994); Kengen et al., FEMS Microbiol. Rev. 18, 119-137 (1996)). Additional energy is generated when excess redox equivalents are channeled to elemental sulfur (Schicho et al., J. Bacteriol. 175, 1823-1830 (1993)).
Most of the proteins isolated from these hyperthermophiles exhibit a temperature optimum of at least 80-100° C. or above (Adams et al., Bio/Technology 13, 662-668 (1995); Adams et al., Trends Biotechnol 16, 329-332 (1998)). Accordingly, there is much interest in exploiting these proteins for biotechnological applications, as they are able to perform biochemical reactions under harsh conditions, such as in the presence of high-temperatures, organic solvents, and denaturants (Adams et al., supra.) P. furiosus has been the source of many of these biotechnologically important proteins, including DNA polymerase (Lundberg et al., Gene 108, 1-6 (1991)), α-amylase (Laderman et al., J. Biol. Chem. 268, 24394-24401 (1993)), and proteases (Voorhorst et al., J. Biol. Chem. 271, 20426-20431 (1996); Harwood et al., J. Bacteriol. 179, 3613-3618 (1997)).
Peptidases hydrolyze peptide bonds from peptide and protein molecules, For example, carboxypeptidases sequentially hydrolyze peptide bonds from the C-terminus of proteins and polypeptides. They are ubiquitous in animals, plants and microorganisms; many carboxypeptidases have been characterized based on their substrate specificity and mechanism (serine- versus metallo-carboxypeptidase) (Skidgel et al., Immunol. Rev. 161, 129-141 (1998)). Carboxypeptidases have been implicated in physiological roles such as protein degradation/turnover, or processing of precursor proteins (Steiner, D. F., Curr. Opin. Chem. Biol. 2, 31-39 (1998)), and in the metabolism of proteins and peptides as carbon or energy sources. Most of the purified carboxypeptidases have a temperature optimum below 40° C. However, three moderately thermostable carboxypeptidases have been purified from the bacteria Thermoactinomyces vulgaris (Stepanov et al., T. Methods Enzymol. 248, 675-683 (1995)), and Thermus aquaticus (Lee et al., Biosci. Biotechnol. Biochem. 56, 1839-1844 (1992)), and the archaeon Sulfolobus solfataricus (Colombo et al., Eur. J. Biochem. 206, 349-357 (1992)), with temperature optima of 60, 80, and 85° C., respectively. Carboxypeptidase activity has not yet been reported for P. furiosus. 
Protein sequencing is an integral component of modern biochemical research. Edman degradation is useful for N-terminal sequencing, but it fails when the amino terminus is chemically protected. Aside from endoproteolytic fragmentation, another way to obtain sequence information from proteins is to sequence from the C-terminus. Various C-terminal sequencing methods have been developed: chemical cleavage analogous to Edman degradation (Hardeman et al., Protein Sci. 7, 1593-1602 (1998)), and enzymatic digestion by carboxypeptidases (Thiede et al., Eur. J. Biochem. 244, 750-754 (1997)). A particularly powerful approach is enzymatic ladder sequencing, in which a carboxypeptidase is used to generate a set of differentially cleaved peptides that can be visualized in a mass spectrum; mass differences between adjacent peaks correspond to the molecular masses of individual amino acids that have been released. Enzymatic protein ladder sequencing has the potential to sequence as far as the enzyme can cut. However, a number of difficulties have limited the applicability of this approach: i) the limited specificities of a given carboxypeptidase toward the 20 common amino acids; and ii) the resistance of native protein molecules to digestion at mesophilic temperatures.
There is a need for novel peptidase enzymes having enhanced thermostability. This includes a need for thermostable C-terminal peptidases whose enhanced thermostability is beneficial in sequencing reactions.